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PinX1 Is a Novel Microtubule-binding Protein Essential for Accurate Chromosome Segregation

2009; Elsevier BV; Volume: 284; Issue: 34 Linguagem: Inglês

10.1074/jbc.m109.001990

1083-351X

Kai Yuan, Na Li, Kai Jiang, Tongge Zhu, Yuda Huo, Chong Wang, Jing Lü, Andrew Shaw, Kelwyn Thomas, Jiancun Zhang, David Mann, Jian Liao, Changjiang Jin, Xuebiao Yao,

Nuclear Structure and Function

Mitosis is an orchestration of dynamic interactions between spindle microtubules and chromosomes, which is mediated by protein structures that include the kinetochores, and other protein complexes present on chromosomes. PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and the outer plate of the kinetochores in mitosis. Our deletion analyses mapped the kinetochore localization domain of PinX1 to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Taken together, the results indicate that PinX1 plays an important role in faithful chromosome segregation in mitosis. Mitosis is an orchestration of dynamic interactions between spindle microtubules and chromosomes, which is mediated by protein structures that include the kinetochores, and other protein complexes present on chromosomes. PinX1 is a potent telomerase inhibitor in interphase; however, its function in mitosis is not well documented. Here we show that PinX1 is essential for faithful chromosome segregation. Deconvolution microscopic analyses show that PinX1 localizes to nucleoli and telomeres in interphase and relocates to the periphery of chromosomes and the outer plate of the kinetochores in mitosis. Our deletion analyses mapped the kinetochore localization domain of PinX1 to the central region and its chromosome periphery localization domain to the C terminus. Interestingly, the kinetochore localization of PinX1 is dependent on Hec1 and CENP-E. Our biochemical characterization revealed that PinX1 is a novel microtubule-binding protein. Our real time imaging analyses show that suppression of PinX1 by small interference RNA abrogates faithful chromosome segregation and results in anaphase chromatid bridges in mitosis and micronuclei in interphase, suggesting an essential role of PinX1 in chromosome stability. Taken together, the results indicate that PinX1 plays an important role in faithful chromosome segregation in mitosis. During mitosis, chromosome movements are orchestrated by the interactions between spindle microtubules and chromosomes. Studies over the last 2 decades have described the kinetochore as the major site where microtubule-chromosome attachment occurs (1Rieder C.L. Int. Rev. Cytol. 1982; 79: 1-58Crossref PubMed Scopus (293) Google Scholar). Electron microscopy has revealed that the kinetochore is composed of four layers as follows: an inner plate, an interzone, an outer plate, and an outermost fibrous corona (2Yao X. Anderson K.L. Cleveland D.W. J. Cell Biol. 1997; 139: 435-447Crossref PubMed Scopus (185) Google Scholar). The outer plate and fibrous corona layers are thought to be the main microtubule-binding sites (1Rieder C.L. Int. Rev. Cytol. 1982; 79: 1-58Crossref PubMed Scopus (293) Google Scholar), and it is known that several protein complexes harboring microtubule binding ability are located in these layers (3Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (304) Google Scholar, 4Cheeseman I.M. Chappie J.S. Wilson-Kubalek E.M. Desai A. Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 5DeLuca J.G. Gall W.E. Ciferri C. Cimini D. Musacchio A. Salmon E.D. Cell. 2006; 127: 969-982Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 6Liu D. Ding X. Du J. Cai X. Huang Y. Ward T. Shaw A. Yang Y. Hu R. Jin C. Yao X. J. Biol. Chem. 2007; 282: 21415-21424Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Wei R.R. Al-Bassam J. Harrison S.C. Nat. Struct. Mol. Biol. 2007; 14: 54-59Crossref PubMed Scopus (254) Google Scholar). Meanwhile, through recruiting several microtubule-dependent motor proteins, the kinetochores generate tension and power chromosome movements in mitosis (6Liu D. Ding X. Du J. Cai X. Huang Y. Ward T. Shaw A. Yang Y. Hu R. Jin C. Yao X. J. Biol. Chem. 2007; 282: 21415-21424Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 8Vergnolle M.A. Taylor S.S. Curr. Biol. 2007; 17: 1173-1179Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Advancements in genomics and proteomics have enabled the identification of additional kinetochore components that are important in governing faithful chromosome segregation (9Foltz D.R. Jansen L.E. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. Nat. Cell Biol. 2006; 8: 458-469Crossref PubMed Scopus (516) Google Scholar, 10Fujita Y. Hayashi T. Kiyomitsu T. Toyoda Y. Kokubu A. Obuse C. Yanagida M. Dev. Cell. 2007; 12: 17-30Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar).PinX1 is a 328-amino acid protein that was originally identified as a Pin2/TRF1 interacting protein in a yeast two-hybrid screen. PinX1 binds to Pin2/TRF1 through its C-terminal 142–254 amino acids. Overexpression of PinX1 or its telomerase inhibitory domain suppresses telomerase activity, causes telomere shortening, and induces cells into crisis, whereas depletion of PinX1 increases telomerase activity and elongates telomeres (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Moreover, PinX1 can directly interact with the human telomerase RNA-binding domain of human telomerase reverse transcriptase as well as human telomerase RNA subunit in vitro (12Banik S.S. Counter C.M. J. Biol. Chem. 2004; 279: 51745-51748Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), suggesting that it acts as an endogenous telomerase inhibitor. Yeast PinX1 inhibits telomerase by sequestering its catalytic subunit in an inactive complex lacking telomerase RNA in nucleoli (13Lin J. Blackburn E.H. Genes Dev. 2004; 18: 387-396Crossref PubMed Scopus (61) Google Scholar). It has been reported that yeast PinX1 is also involved in rRNA and small nucleolar RNA maturation (14Guglielmi B. Werner M. J. Biol. Chem. 2002; 277: 35712-35719Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The rat homolog of PinX1 also localizes to nucleoli in interphase and regulates telomere length (15Oh B.K. Yoon S.M. Lee C.H. Park Y.N. Gene. 2007; 400: 35-43Crossref PubMed Scopus (11) Google Scholar). In human cells, it is reported that PinX1 has an effect on mediating human telomerase reverse transcriptase nucleolar localization (16Lin J. Jin R. Zhang B. Yang P.X. Chen H. Bai Y.X. Xie Y. Huang C. Huang J. Biochem. Biophys. Res. Commun. 2007; 353: 946-952Crossref PubMed Scopus (21) Google Scholar). Collectively, these studies demonstrate that the functions of PinX1 in cell growth regulation are well conserved during evolution. Indeed, loss of heterozygosity of PinX1 occurs at a high frequency in many human cancers (17Wright K. Wilson P.J. Kerr J. Do K. Hurst T. Khoo S.K. Ward B. Chenevix-Trench G. Oncogene. 1998; 17: 1185-1188Crossref PubMed Scopus (62) Google Scholar), and animal studies showed that depletion of endogenous PinX1 promotes tumorigenicity in nude mice (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar).As described above, the localization of PinX1 in interphase and its role in regulating telomere length have been well investigated. However, it has remained elusive as to whether PinX1 plays any role in mitosis and what happens if PinX1 is deficient. In this study, we have demonstrated that PinX1 is localized to the outer plate of kinetochores during mitosis. PinX1 is essential for spindle stability because depletion of PinX1 in HeLa cells destabilizes kinetochore microtubules and results in lagging chromosomes. Importantly, PinX1 interacts with microtubules. Our functional analyses show that PinX1 plays an important role in governing chromosome segregation and genomic stability.DISCUSSIONAneuploidy, a loss or gain of chromosomes, a major form of chromosome instability commonly associated with cancer formation and progression, is thought to arise from aberrant mitotic chromosome segregation. Chromosome segregation in mitosis is orchestrated by the interactions of kinetochores with spindle microtubules and is monitored by the spindle assembly checkpoint proteins (28Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1702) Google Scholar). In this study, we have demonstrated that PinX1 localizes to the chromosome periphery during mitosis. In addition, we revealed the kinetochore localization of PinX1, which persists from prophase to metaphase, suggesting that PinX1 may be involved in regulating chromosome movements. Suppression of the expression of PinX1 by siRNA in HeLa cells results in depletion of PinX1 during mitosis, destabilization of kinetochore microtubules, and chromosome instability.Besides its kinetochore and chromosome periphery localization, exogenously expressed GFP-PinX1 was readily apparent on the spindle poles. However, the spindle pole-associated endogenous PinX1 was less prominent in our experimental condition. It is possible that the PinX1 epitope recognized by our antibody is somehow masked at the spindle pole. The apparent spindle pole-associated localization of GFP-PinX1 could also be due to the fact that overexpression of GFP-PinX1 saturates its binding sites at the kinetochore and chromosome periphery leading to accumulation at the poles. Nevertheless, it is worth noting that TRF1, a binding partner of PinX1, is redistributed to the spindle poles in mitosis (29Nakamura M. Zhou X.Z. Kishi S. Kosugi I. Tsutsui Y. Lu K.P. Curr. Biol. 2001; 11: 1512-1516Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, bipolar spindle integrity was not altered in PinX1-depleted cells, suggesting the mitotic defects seen in the PinX1 suppression are not associated with its spindle localization. Thus, we reason that the function of PinX1 in mitosis is mainly tied to its localization on the kinetochores.It was surprising that PinX1 bears microtubule binding activity. However, our attempt to map the microtubule binding activity using PinX1 deletion mutants was unsuccessful. It is possible that deletion disrupted the secondary structure necessary for the microtubule binding. Given the importance of PinX1 in mitotic chromosome segregation, it would be of great importance to characterize its structure-function relationship. Chromosome instability phenotype seen in PinX1-repressed cells is consistent with the fact that depletion of PinX1 also increases tumorigenicity in nude mice (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar).Recent studies have revealed that other structures on the chromosomes facilitate faithful chromosome segregation (30Ma N. Matsunaga S. Takata H. Ono-Maniwa R. Uchiyama S. Fukui K. J. Cell Sci. 2007; 120: 2091-2105Crossref PubMed Scopus (102) Google Scholar, 31Sakita-Suto S. Kanda A. Suzuki F. Sato S. Takata T. Tatsuka M. Mol. Biol. Cell. 2007; 18: 1107-1117Crossref PubMed Scopus (63) Google Scholar). Proteomic analysis of mitotic chromosomes identified hundreds of chromosome periphery proteins in addition to the kinetochore proteins (32Uchiyama S. Kobayashi S. Takata H. Ishihara T. Hori N. Higashi T. Hayashihara K. Sone T. Higo D. Nirasawa T. Takao T. Matsunaga S. Fukui K. J. Biol. Chem. 2005; 280: 16994-17004Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The large number of proteins observed in perichromosomal regions during mitosis implies that they play functional roles in this process (33Van Hooser A.A. Yuh P. Heald R. Chromosoma. 2005; 114: 377-388Crossref PubMed Scopus (73) Google Scholar). In fact, Heald et al. (34Heald R. Tournebize R. Blank T. Sandaltzopoulos R. Becker P. Hyman A. Karsenti E. Nature. 1996; 382: 420-425Crossref PubMed Scopus (774) Google Scholar) have elegantly demonstrated that bipolar spindles can be assembled spontaneously around artificial chromosomes using Xenopus egg extracts, suggesting a role of chromosomes, perhaps chromosome periphery proteins, in microtubule nucleation and spindle assembly. Subsequent investigation identified a Rae1-containing ribonucleoprotein complex that is involved in spindle assembly and particularly in microtubule nucleation and stabilization (35Blower M.D. Nachury M. Heald R. Weis K. Cell. 2005; 121: 223-234Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Several studies demonstrate that Ran-GTP gradients generated by the chromosomal Ran-guanine nucleotide exchange factor RCC1 are essential for chromosome-mediated bipolar spindle assembly (36Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 37Kalab P. Weis K. Heald R. Science. 2002; 295: 2452-2456Crossref PubMed Scopus (437) Google Scholar). More recently, the Ran-importin-β complex was reported to be involved in chromosome loading of human chromokinesin Kid (38Tahara K. Takagi M. Ohsugi M. Sone T. Nishiumi F. Maeshima K. Horiuchi Y. Tokai-Nishizumi N. Imamoto F. Yamamoto T. Kose S. Imamoto N. J. Cell Biol. 2008; 180: 493-506Crossref PubMed Scopus (45) Google Scholar). Chromokinesins generate polar ejection forces on chromosomes to push them toward the microtubule plus-end and thus the equatorial plane (39Mazumdar M. Misteli T. Trends Cell Biol. 2005; 15: 349-355Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Considering the chromosome congression defect observed in PinX1-depleted cells, it will be worth verifying whether PinX1 is involved in chromosome loading of chromokinesins or the generation of Ran-GTP gradients.Taken together, our findings demonstrate a critical role of PinX1 in accurate chromosome segregation. The fact that elimination of PinX1 disrupts kinetochore microtubule-kinetochore association and induces chromosome cross-bridges in HeLa cells prematurely exited from anaphase demonstrates the importance of PinX1-microtubule interaction in facilitating chromosome segregation and the maintenance of genomic stability in mitosis. During mitosis, chromosome movements are orchestrated by the interactions between spindle microtubules and chromosomes. Studies over the last 2 decades have described the kinetochore as the major site where microtubule-chromosome attachment occurs (1Rieder C.L. Int. Rev. Cytol. 1982; 79: 1-58Crossref PubMed Scopus (293) Google Scholar). Electron microscopy has revealed that the kinetochore is composed of four layers as follows: an inner plate, an interzone, an outer plate, and an outermost fibrous corona (2Yao X. Anderson K.L. Cleveland D.W. J. Cell Biol. 1997; 139: 435-447Crossref PubMed Scopus (185) Google Scholar). The outer plate and fibrous corona layers are thought to be the main microtubule-binding sites (1Rieder C.L. Int. Rev. Cytol. 1982; 79: 1-58Crossref PubMed Scopus (293) Google Scholar), and it is known that several protein complexes harboring microtubule binding ability are located in these layers (3Yao X. Abrieu A. Zheng Y. Sullivan K.F. Cleveland D.W. Nat. Cell Biol. 2000; 2: 484-491Crossref PubMed Scopus (304) Google Scholar, 4Cheeseman I.M. Chappie J.S. Wilson-Kubalek E.M. Desai A. Cell. 2006; 127: 983-997Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 5DeLuca J.G. Gall W.E. Ciferri C. Cimini D. Musacchio A. Salmon E.D. Cell. 2006; 127: 969-982Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 6Liu D. Ding X. Du J. Cai X. Huang Y. Ward T. Shaw A. Yang Y. Hu R. Jin C. Yao X. J. Biol. Chem. 2007; 282: 21415-21424Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Wei R.R. Al-Bassam J. Harrison S.C. Nat. Struct. Mol. Biol. 2007; 14: 54-59Crossref PubMed Scopus (254) Google Scholar). Meanwhile, through recruiting several microtubule-dependent motor proteins, the kinetochores generate tension and power chromosome movements in mitosis (6Liu D. Ding X. Du J. Cai X. Huang Y. Ward T. Shaw A. Yang Y. Hu R. Jin C. Yao X. J. Biol. Chem. 2007; 282: 21415-21424Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 8Vergnolle M.A. Taylor S.S. Curr. Biol. 2007; 17: 1173-1179Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Advancements in genomics and proteomics have enabled the identification of additional kinetochore components that are important in governing faithful chromosome segregation (9Foltz D.R. Jansen L.E. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. Nat. Cell Biol. 2006; 8: 458-469Crossref PubMed Scopus (516) Google Scholar, 10Fujita Y. Hayashi T. Kiyomitsu T. Toyoda Y. Kokubu A. Obuse C. Yanagida M. Dev. Cell. 2007; 12: 17-30Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). PinX1 is a 328-amino acid protein that was originally identified as a Pin2/TRF1 interacting protein in a yeast two-hybrid screen. PinX1 binds to Pin2/TRF1 through its C-terminal 142–254 amino acids. Overexpression of PinX1 or its telomerase inhibitory domain suppresses telomerase activity, causes telomere shortening, and induces cells into crisis, whereas depletion of PinX1 increases telomerase activity and elongates telomeres (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Moreover, PinX1 can directly interact with the human telomerase RNA-binding domain of human telomerase reverse transcriptase as well as human telomerase RNA subunit in vitro (12Banik S.S. Counter C.M. J. Biol. Chem. 2004; 279: 51745-51748Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), suggesting that it acts as an endogenous telomerase inhibitor. Yeast PinX1 inhibits telomerase by sequestering its catalytic subunit in an inactive complex lacking telomerase RNA in nucleoli (13Lin J. Blackburn E.H. Genes Dev. 2004; 18: 387-396Crossref PubMed Scopus (61) Google Scholar). It has been reported that yeast PinX1 is also involved in rRNA and small nucleolar RNA maturation (14Guglielmi B. Werner M. J. Biol. Chem. 2002; 277: 35712-35719Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The rat homolog of PinX1 also localizes to nucleoli in interphase and regulates telomere length (15Oh B.K. Yoon S.M. Lee C.H. Park Y.N. Gene. 2007; 400: 35-43Crossref PubMed Scopus (11) Google Scholar). In human cells, it is reported that PinX1 has an effect on mediating human telomerase reverse transcriptase nucleolar localization (16Lin J. Jin R. Zhang B. Yang P.X. Chen H. Bai Y.X. Xie Y. Huang C. Huang J. Biochem. Biophys. Res. Commun. 2007; 353: 946-952Crossref PubMed Scopus (21) Google Scholar). Collectively, these studies demonstrate that the functions of PinX1 in cell growth regulation are well conserved during evolution. Indeed, loss of heterozygosity of PinX1 occurs at a high frequency in many human cancers (17Wright K. Wilson P.J. Kerr J. Do K. Hurst T. Khoo S.K. Ward B. Chenevix-Trench G. Oncogene. 1998; 17: 1185-1188Crossref PubMed Scopus (62) Google Scholar), and animal studies showed that depletion of endogenous PinX1 promotes tumorigenicity in nude mice (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). As described above, the localization of PinX1 in interphase and its role in regulating telomere length have been well investigated. However, it has remained elusive as to whether PinX1 plays any role in mitosis and what happens if PinX1 is deficient. In this study, we have demonstrated that PinX1 is localized to the outer plate of kinetochores during mitosis. PinX1 is essential for spindle stability because depletion of PinX1 in HeLa cells destabilizes kinetochore microtubules and results in lagging chromosomes. Importantly, PinX1 interacts with microtubules. Our functional analyses show that PinX1 plays an important role in governing chromosome segregation and genomic stability. DISCUSSIONAneuploidy, a loss or gain of chromosomes, a major form of chromosome instability commonly associated with cancer formation and progression, is thought to arise from aberrant mitotic chromosome segregation. Chromosome segregation in mitosis is orchestrated by the interactions of kinetochores with spindle microtubules and is monitored by the spindle assembly checkpoint proteins (28Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1702) Google Scholar). In this study, we have demonstrated that PinX1 localizes to the chromosome periphery during mitosis. In addition, we revealed the kinetochore localization of PinX1, which persists from prophase to metaphase, suggesting that PinX1 may be involved in regulating chromosome movements. Suppression of the expression of PinX1 by siRNA in HeLa cells results in depletion of PinX1 during mitosis, destabilization of kinetochore microtubules, and chromosome instability.Besides its kinetochore and chromosome periphery localization, exogenously expressed GFP-PinX1 was readily apparent on the spindle poles. However, the spindle pole-associated endogenous PinX1 was less prominent in our experimental condition. It is possible that the PinX1 epitope recognized by our antibody is somehow masked at the spindle pole. The apparent spindle pole-associated localization of GFP-PinX1 could also be due to the fact that overexpression of GFP-PinX1 saturates its binding sites at the kinetochore and chromosome periphery leading to accumulation at the poles. Nevertheless, it is worth noting that TRF1, a binding partner of PinX1, is redistributed to the spindle poles in mitosis (29Nakamura M. Zhou X.Z. Kishi S. Kosugi I. Tsutsui Y. Lu K.P. Curr. Biol. 2001; 11: 1512-1516Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, bipolar spindle integrity was not altered in PinX1-depleted cells, suggesting the mitotic defects seen in the PinX1 suppression are not associated with its spindle localization. Thus, we reason that the function of PinX1 in mitosis is mainly tied to its localization on the kinetochores.It was surprising that PinX1 bears microtubule binding activity. However, our attempt to map the microtubule binding activity using PinX1 deletion mutants was unsuccessful. It is possible that deletion disrupted the secondary structure necessary for the microtubule binding. Given the importance of PinX1 in mitotic chromosome segregation, it would be of great importance to characterize its structure-function relationship. Chromosome instability phenotype seen in PinX1-repressed cells is consistent with the fact that depletion of PinX1 also increases tumorigenicity in nude mice (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar).Recent studies have revealed that other structures on the chromosomes facilitate faithful chromosome segregation (30Ma N. Matsunaga S. Takata H. Ono-Maniwa R. Uchiyama S. Fukui K. J. Cell Sci. 2007; 120: 2091-2105Crossref PubMed Scopus (102) Google Scholar, 31Sakita-Suto S. Kanda A. Suzuki F. Sato S. Takata T. Tatsuka M. Mol. Biol. Cell. 2007; 18: 1107-1117Crossref PubMed Scopus (63) Google Scholar). Proteomic analysis of mitotic chromosomes identified hundreds of chromosome periphery proteins in addition to the kinetochore proteins (32Uchiyama S. Kobayashi S. Takata H. Ishihara T. Hori N. Higashi T. Hayashihara K. Sone T. Higo D. Nirasawa T. Takao T. Matsunaga S. Fukui K. J. Biol. Chem. 2005; 280: 16994-17004Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The large number of proteins observed in perichromosomal regions during mitosis implies that they play functional roles in this process (33Van Hooser A.A. Yuh P. Heald R. Chromosoma. 2005; 114: 377-388Crossref PubMed Scopus (73) Google Scholar). In fact, Heald et al. (34Heald R. Tournebize R. Blank T. Sandaltzopoulos R. Becker P. Hyman A. Karsenti E. Nature. 1996; 382: 420-425Crossref PubMed Scopus (774) Google Scholar) have elegantly demonstrated that bipolar spindles can be assembled spontaneously around artificial chromosomes using Xenopus egg extracts, suggesting a role of chromosomes, perhaps chromosome periphery proteins, in microtubule nucleation and spindle assembly. Subsequent investigation identified a Rae1-containing ribonucleoprotein complex that is involved in spindle assembly and particularly in microtubule nucleation and stabilization (35Blower M.D. Nachury M. Heald R. Weis K. Cell. 2005; 121: 223-234Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Several studies demonstrate that Ran-GTP gradients generated by the chromosomal Ran-guanine nucleotide exchange factor RCC1 are essential for chromosome-mediated bipolar spindle assembly (36Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. Cell. 2001; 104: 95-106Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 37Kalab P. Weis K. Heald R. Science. 2002; 295: 2452-2456Crossref PubMed Scopus (437) Google Scholar). More recently, the Ran-importin-β complex was reported to be involved in chromosome loading of human chromokinesin Kid (38Tahara K. Takagi M. Ohsugi M. Sone T. Nishiumi F. Maeshima K. Horiuchi Y. Tokai-Nishizumi N. Imamoto F. Yamamoto T. Kose S. Imamoto N. J. Cell Biol. 2008; 180: 493-506Crossref PubMed Scopus (45) Google Scholar). Chromokinesins generate polar ejection forces on chromosomes to push them toward the microtubule plus-end and thus the equatorial plane (39Mazumdar M. Misteli T. Trends Cell Biol. 2005; 15: 349-355Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Considering the chromosome congression defect observed in PinX1-depleted cells, it will be worth verifying whether PinX1 is involved in chromosome loading of chromokinesins or the generation of Ran-GTP gradients.Taken together, our findings demonstrate a critical role of PinX1 in accurate chromosome segregation. The fact that elimination of PinX1 disrupts kinetochore microtubule-kinetochore association and induces chromosome cross-bridges in HeLa cells prematurely exited from anaphase demonstrates the importance of PinX1-microtubule interaction in facilitating chromosome segregation and the maintenance of genomic stability in mitosis. Aneuploidy, a loss or gain of chromosomes, a major form of chromosome instability commonly associated with cancer formation and progression, is thought to arise from aberrant mitotic chromosome segregation. Chromosome segregation in mitosis is orchestrated by the interactions of kinetochores with spindle microtubules and is monitored by the spindle assembly checkpoint proteins (28Musacchio A. Salmon E.D. Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1702) Google Scholar). In this study, we have demonstrated that PinX1 localizes to the chromosome periphery during mitosis. In addition, we revealed the kinetochore localization of PinX1, which persists from prophase to metaphase, suggesting that PinX1 may be involved in regulating chromosome movements. Suppression of the expression of PinX1 by siRNA in HeLa cells results in depletion of PinX1 during mitosis, destabilization of kinetochore microtubules, and chromosome instability. Besides its kinetochore and chromosome periphery localization, exogenously expressed GFP-PinX1 was readily apparent on the spindle poles. However, the spindle pole-associated endogenous PinX1 was less prominent in our experimental condition. It is possible that the PinX1 epitope recognized by our antibody is somehow masked at the spindle pole. The apparent spindle pole-associated localization of GFP-PinX1 could also be due to the fact that overexpression of GFP-PinX1 saturates its binding sites at the kinetochore and chromosome periphery leading to accumulation at the poles. Nevertheless, it is worth noting that TRF1, a binding partner of PinX1, is redistributed to the spindle poles in mitosis (29Nakamura M. Zhou X.Z. Kishi S. Kosugi I. Tsutsui Y. Lu K.P. Curr. Biol. 2001; 11: 1512-1516Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, bipolar spindle integrity was not altered in PinX1-depleted cells, suggesting the mitotic defects seen in the PinX1 suppression are not associated with its spindle localization. Thus, we reason that the function of PinX1 in mitosis is mainly tied to its localization on the kinetochores. It was surprising that PinX1 bears microtubule binding activity. However, our attempt to map the microtubule binding activity using PinX1 deletion mutants was unsuccessful. It is possible that deletion disrupted the secondary structure necessary for the microtubule binding. Given the importance of PinX1 in mitotic chromosome segregation, it would be of great importance to characterize its structure-function relationship. Chromosome instability phenotype seen in PinX1-repressed cells is consistent with the fact that depletion of PinX1 also increases tumorigenicity in nude mice (11Zhou X.Z. Lu K.P. Cell. 2001; 107: 347-359Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Recent studies have revealed that other structures on the chromosomes facilitate faithful chromosome segregation (30Ma N. Matsunaga S. Takata H. Ono-Maniwa R. Uchiyama S. Fukui K. J. Cell Sci. 2007; 120: 2091-2105Crossref PubMed Scopus (102) Google Scholar, 31Sakita-Suto S. Kanda A. Suzuki F. Sato S. Takata T. Tatsuka M. Mol. Biol. Cell. 2007; 18: 1107-1117Crossref PubMed Scopus (63) Google Scholar). Proteomic analysis of mitotic chromosomes identified hundreds of chromosome periphery proteins in addition to the kinetochore proteins (32Uchiyama S. Kobayashi S. Takata H. Ishihara T. Hori N. Higashi T. Hayashihara K. Sone T. Higo D. Nirasawa T. Takao T. Matsunaga S. Fukui K. J. Biol. Chem. 2005; 280: 16994-17004Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The large number of proteins observed in perichromosomal regions during mitosis implies that they play functional roles in this process (33Van Hooser A.A. Yuh P. Heald R. Chromosoma. 2005; 114: 377-388Crossref PubMed Scopus (73) Google Scholar). In fact, Heald et al. (34Heald R. Tournebize R. Blank T. Sandaltzopoulos R. Becker P. Hyman A. Karsenti E. Nature. 1996; 382: 420-425Crossref PubMed Scopus (774) Google Scholar) have elegantly demonstrated that bipolar spindles can be assembled spontaneously around artificial chromosomes using Xenopus egg extracts, suggesting a role of chromosomes, perhaps chromosome periphery proteins, in microtubule nucleation and spindle assembly. Subsequent investigation identified a Rae1-containing ribonucleoprotein complex that is involved in spindle assembly and particularly in microtubule nucleation and stabilization (35Blower M.D. Nachury M. Heald R. Weis K. Cell. 2005; 121: 223-234Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Several studies demonstrate that Ran-GTP gradients generated by the chromosomal Ran-guanine nucleotide exchange factor RCC1 are essential for chromosome-mediated bipolar spindle assembly (36Nachury M.V. Maresca T.J. Salmon W.C. Waterman-Storer C.M. Heald R. Weis K. 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Considering the chromosome congression defect observed in PinX1-depleted cells, it will be worth verifying whether PinX1 is involved in chromosome loading of chromokinesins or the generation of Ran-GTP gradients. Taken together, our findings demonstrate a critical role of PinX1 in accurate chromosome segregation. The fact that elimination of PinX1 disrupts kinetochore microtubule-kinetochore association and induces chromosome cross-bridges in HeLa cells prematurely exited from anaphase demonstrates the importance of PinX1-microtubule interaction in facilitating chromosome segregation and the maintenance of genomic stability in mitosis. We thank Dr. Kunping Lu, Dr. Nancy Maizel, and Dr. Guowei Fang for reagents. The facilities used were supported in part by National Institutes of Health Grant G-12-RR03034 from NCRR/RCM1. Supplementary Material Download .pdf (.3 MB) Help with pdf files Download .pdf (.3 MB) Help with pdf files